Compositions comprising ch505 envelopes, and trimers

ABSTRACT

In certain aspects the invention provides a selection of HIV-1 envelopes suitable for use as immunogens, and methods of using these immunogens to induce neutralizing antibodies. In certain embodiments, the immunogens are designed to trimerize. In other embodiments, the immunogens comprise an immune modulating component.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to the U.S.Provisional Patent Application No. 62/056,602, entitled “CompositionsComprising CH505 Envelopes, and Trimers” filed on Sep. 28, 2014, thecontents of each of which are hereby incorporated by reference in theirentirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under Center forHIV/AIDS Vaccine Immunology-Immunogen Design grant UM1-A1100645 from theNIH, NIAID, Division of AIDS. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates in general, to a composition suitable foruse in inducing anti-HIV-1 antibodies, and, in particular, toimmunogenic compositions comprising envelope proteins and nucleic acidsto induce cross-reactive neutralizing antibodies and increase theirbreadth of coverage. The invention also relates to methods of inducingsuch broadly neutralizing anti-HIV-1 antibodies using such compositions.

BACKGROUND

The development of a safe and effective HIV-1 vaccine is one of thehighest priorities of the scientific community working on the HIV-1epidemic. While anti-retroviral treatment (ART) has dramaticallyprolonged the lives of HIV-1 infected patients, ART is not routinelyavailable in developing countries.

SUMMARY OF THE INVENTION

In certain aspects the invention is directed to HIV-1 envelopes whichare designed as fusion molecules comprising a portion of an envelopeprotein and a trimerization domain so as to trimerize. In certainaspects the invention is directed towards methods of using such HIV-1envelopes for immunization so as to induce immune response, whichcomprises humoral immune response. In certain embodiments, the methodsof immunization comprise administering an agent which transientlymodulates the immune response.

In certain aspects, the invention provides a composition comprising anyone of the HIV-1 envelope polypeptides corresponding to the HIV-1envelopes CH505.M6, CH505.M11, CH505w020.14, CH505w030.28, CH505w078.15,CH505w053.31, CH505w030.21, CH505w078.33, CH505w053.16, CH505w100.B6 ora combination thereof, or polynucleotide encoding the same.

In certain aspects, the invention provides a composition comprising anyone of the HIV-1 envelope polypeptides corresponding to the HIV-1envelopes CH505.T/F; CH505.M11; CH505w020.14; CH505w030.28;CH505w030.21; CH505w053.16; CH505w053.31; CH505w078.33; CH505w078.15;CH505w100.B6 or a combination thereof, or a polynucleotide encoding thesame.

In certain aspects, the invention provides a composition comprising anyone of the HIV-1 envelope polypeptides corresponding to the HIV-1envelopes CH505.M11, CH505.w004.03, CH505.w020.14, CH505.w030.28,CH505.w030.12, CH505.w020.2, CH505.w030.10, CH505.w078.15, CH505.w030.19CH505.w030.21 or a combination thereof, or polynucleotide encoding thesame.

In certain aspects the invention provides, a composition comprising anyone of the polynucleotides in Table 4 or Table 5 or a combinationthereof. In some embodiments the compositions comprises envelopeproteins. In some embodiments, the composition comprises polynucleotidesencoding the envelope proteins.

In certain embodiments, the proteins or polynucleotides comprise atrimerization domain. In certain embodiments the trimerziation domain isGCN4. In certain embodiments the trimerization domain is CD40L. Incertain embodiments the trimerization domain is linked to the envelopesequence via a linker. In certain embodiments the linker is about 6amino acids. In other embodiments the linker is about 3-20 amino acids.In certain embodiments, the linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 amino acids.

In certain embodiments, the proteins or polynucleotides furthercomprises a CD40L sequence.

In certain aspects the invention provides a composition comprisingMPER-peptide-liposome-CD40L conjugate.

In certain embodiments, the compositions of the invention furthercomprise an adjuvant.

In one aspect the invention provides methods of inducing an immuneresponse in a subject comprising administering the composition of anyone of the compositions in an amount sufficient to induce an immuneresponse.

In certain embodiments, the methods comprise administering any suitableagent or transient immune modulation which could modulate mechanisms ofhost immune tolerance and release of the induced antibodies. In certainembodiments, the methods of the invention comprise administeringchloloquine before each immunization. In certain embodiments,chloloquine is administered for about 10 days before each immunization.

In certain embodiments, the methods of the invention compriseadministering anti-CD25 antibody prior or after each immunization. Incertain embodiments, anti-CD25 antibody is administered for about 5 daysbefore each immunization.

In certain embodiments, the methods of the invention comprisecompositions which comprise a nucleic acid, a protein or any combinationthereof. In certain embodiments, the nucleic acid encoding the envelopeis operably linked to a promoter inserted in an expression vector. Incertain embodiments, the protein is recombinant.

In certain aspects, the invention provides a composition comprising anyone of the HIV-1 envelope polypeptides corresponding to the HIV-1envelopes 703010505.TF, 703010505.W53.16, 703010505.W78.33,703010505.W100.B6 or a combination thereof, or polynucleotide encodingthe same.

In certain aspects, the invention provides a composition comprising anyone of the HIV-1 envelope polypeptides corresponding to the HIV-1envelopes 703010505.TF, 703010505.W4.03, 703010505.W4.26,703010505.W14.21, 703010505.W20.14, 703010505.W30.28, 703010505.W30.13,703010505.W53.31, 703010505.W78.15, 703010505.W100.B4 or a combinationthereof, or polynucleotide encoding the same.

In certain aspects, the invention provides a composition comprising anyone of the HIV-1 envelope polypeptides corresponding to the HIV-1envelopes 703010505.TF, 703010505.W4.03, 703010505.W4.26,703010505.W14.3, 703010505.W14.8, 703010505.W14.21, 703010505.W20.7,703010505.W20.26, 703010505.W20.9, 703010505.W20.14, 703010505.W30.28,703010505.W30.12, 703010505.W30.19, 703010505.W30.13, 703010505.W53.19,703010505.W53.13, 703010505.W53.31, 703010505.W78.1, 703010505.W78.15,703010505.W100.B4 or a combination thereof, or polynucleotide encodingthe same.

Also disclosed are compositions wherein each HIV-1 envelope polypeptidecomprises polypeptide or polynucleotide which is a gp 41, gp 120, gp145, gp140, gp 150 or gp 160 variant.

In certain embodiments, the HIV-1 envelopes are administered as anucleic acid, a protein or any combination thereof. In certainembodiments, the nucleic acid encoding the envelope is operably linkedto a promoter inserted in an expression vector. In certain embodiments,the protein is recombinant.

In certain embodiments, the envelopes are administered as a prime, aboost, or both. In certain embodiments, the envelopes, or anycombinations thereof are administered as a multiple boosts. In certainembodiments, the compositions and method further comprise an adjuvant.In certain embodiments, the HIV-1 envelopes are provided as nucleic acidsequences, including but not limited to nucleic acids optimized forexpression in the desired vector and/or host cell. In other embodiments,the HIV-1 envelopes are provided as recombinantly expressed protein.

In certain embodiments, the invention provides compositions and methodfor induction of immune response, for example cross-reactive (broadly)neutralizing Ab induction. In certain embodiments, the methods usecompositions comprising “swarms” of sequentially evolved envelopeviruses that occur in the setting of bnAb generation in vivo in HIV-1infection.

In certain aspects the invention provides compositions comprising aselection of HIV-1 envelopes or nucleic acids encoding these envelopes,for example but not limited to, as described herein. In certainembodiments, these compositions are used in immunization methods as aprime and/or boost, for example but not limited to, as described herein.

In certain embodiments, the compositions contemplate nucleic acid, asDNA and/or RNA, or protein immunogens either alone or in anycombination. In certain embodiments, the methods contemplate genetic, asDNA and/or RNA, immunization either alone or in combination withenvelope protein(s).

In certain embodiments, the nucleic acid encoding an envelope isoperably linked to a promoter inserted in an expression vector. Incertain aspects the compositions comprise a suitable carrier. In certainaspects, the compositions comprise a suitable adjuvant.

In certain embodiments, the induced immune response includes inductionof antibodies, including, but not limited to autologous and/orcross-reactive (broadly) neutralizing antibodies against HIV-1 envelope.Various assays that analyze whether an immunogenic composition inducesan immune response, and the type of antibodies induced are known in theart and are also described herein.

In certain aspects, the invention provides an expression vectorcomprising any of the nucleic acid sequences of the invention, whereinthe nucleic acid is operably linked to a promoter. In certain aspectsthe invention provides an expression vector comprising a nucleic acidsequence encoding any of the polypeptides of the invention, wherein thenucleic acid is operably linked to a promoter. In certain embodiments,the nucleic acids are codon optimized for expression in a mammaliancell, in vivo or in vitro. In certain aspects, the invention providesnucleic acid comprising any one of the nucleic acid sequences ofinvention. Also provided is a nucleic acid consisting essentially of anyone of the nucleic acid sequences of invention. Also provided is anucleic acid consisting of any one of the nucleic acid sequences ofinvention. In certain embodiments, the nucleic acid of invention, isoperably linked to a promoter and is inserted in an expression vector.In certain aspects the invention provides an immunogenic compositioncomprising the expression vector.

In certain aspects, the invention provides a composition comprising atleast one of the nucleic acid sequences of the invention. In certainaspects, the invention provides a composition comprising any one of thenucleic acid sequences of invention. In certain aspects, the inventionprovides a composition comprising a combination of one nucleic acidsequence encoding any one of the polypeptides of the invention. Incertain embodiments, combining DNA and protein gives higher magnitude ofab responses. See Pissani F. Vaccine 32: 507-13, 2013; Jalah R et alPLoS One 9: e91550, 2014.

In certain embodiments, the compositions and methods employ an HIV-1envelope as polypeptide instead of a nucleic acid sequence encoding theHIV-1 envelope. In certain embodiments, the compositions and methodsemploy an HIV-1 envelope as polypeptide, a nucleic acid sequenceencoding the HIV-1 envelope, or a combination thereof. The envelope canbe a gp160, gp150, gp145, gp140, gp120, gp41, N-terminal deletionvariants as described herein, cleavage resistant variants as describedherein, or codon optimized sequences thereof. The polypeptide of theinventions can be a trimer. The polypeptide contemplated by theinvention can be a polypeptide comprising any one of the polypeptidesdescribed herein. The polypeptide contemplated by the invention can be apolypeptide consisting essentially of any one of the polypeptidesdescribed herein. The polypeptide contemplated by the invention can be apolypeptide consisting of any one of the polypeptides described herein.In certain embodiments, the polypeptide is recombinantly produced. Incertain embodiments, the polypeptides and nucleic acids of the inventionare suitable for use as an immunogen, for example to be administered ina human subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows designs of HIV-1 envelopes with trimerization domain, andimmune modulating (e.g. CD40L) domain.

FIGS. 2A and 2B show Envelope monomer timer QC—Non-reducing conditions.

FIGS. 3A and 3B show Envelope monomer timer QC—reducing conditions.

FIG. 4 shows Envelope trimer by Blue Native PAGE.

FIG. 5 shows a summary of the data in FIGS. 6-11. FIG. 5 shows thatCH505TF gp120 GCN4 Trimer binds to CH103 UCA (CD4bs) with a lower Kd(nm) compared to the CH505TF gp120 D7 Monomer.

FIG. 6 shows gp120 trimers antigenicity.

FIG. 7 shows gp120 trimers antigenicity.

FIG. 8 shows gp120 trimers antigenicity.

FIG. 9 shows gp120 trimers antigenicity.

FIG. 10 shows gp120 trimers antigenicity.

FIG. 11 shows gp120 trimers antigenicity.

FIG. 12 shows the design of CD40L-MPER656 peptide-liposome conjugate.

FIG. 13 shows antigenicity of the MPER-liposome of FIG. 12. Biolayerinterferometry assay of binding of mouse anti-human CD40L mAb (A) andbroadly neutralizing HIV-1 gp41 MPER mAbs 2F5 (B) and 4E10 mAbs (C) at20 μg/ml to CD40L-MPER656 liposomes loaded onto Aminopropyl silanesensors are shown. The binding of antibodies to appropriate controlliposomes were subtracted to obtain the specific binding shown in panelA-C.

FIG. 14 show that the CD40L-MPER656 peptide-liposome conjugate isfunctional.

FIG. 15 shows that CH505 gp120-GCN4-CD40L activates human CD40expressing HEK cells.

FIG. 16 shows antigenicity of CH505 gp120-GCN4-CD40L. SPR binding assayessentially as described in FIG. 13.

FIG. 17 shows sequence of a selection of ten envelopes (“P10” derivedfrom CH505).

FIG. 18A shows binding of CH103 antibodies to the autologous Envs ofFIG. 17, log AUC. FIG. 18B shows the selection of 10 CH505 Envs (10 PR)based on the binding intensity for the CH103 members. Humanized mice areimmunized with this selection of HIV-1 envelopes.

FIG. 19 shows neutralization IC50s of CH103 lineage mAbs againstautologous CH505 Envs. Pseudoviruses are sorted by sensitivity toCH103-lineage mAbs, then geometric mean IC50. Here only 108 viruses withdistinct gp120s are shown, not the full set of 135 Envs assayed.

FIG. 20 shows autologous neutralization profiles for (A) mutated TFviruses, (B) 4-Env immunogen set, (C) previously identified 10-Envimmunogen set, and (D) currently identified 10-Env immunogen set.Concatenated sites listed in Table 1 are shown for each candidateimmunogen

FIG. 21 CH505 (A) Env Mutations, (B) CH103 lineage MAb IC50s, and (C)Env phylogeny. Env immunogens proposed in alternative vaccinationregimes are shown by colored diamonds. Unlike earlier phylogenies ofthese Envs, insertions and deletions here are treated as distinctcharacters, rather than missing data.

FIG. 22 show the sequences of four envelopes, grouped as HIV-1 EnvelopesSelection C.

FIG. 23 shows the sequences of CAP 206 envelopes from 6 months.

FIG. 24 shows the sequence of ten early envelopes, grouped as HIV-1Envelopes Selection E.

DETAILED DESCRIPTION

The development of a safe, highly efficacious prophylactic HIV-1 vaccineis of paramount importance for the control and prevention of HIV-1infection. A major goal of HIV-1 vaccine development is the induction ofbroadly neutralizing antibodies (bnAbs) (Immunol. Rev. 254: 225-244,2013). BnAbs are protective in rhesus macaques against SHIV challenge,but as yet, are not induced by current vaccines.

For the past 25 years, the HIV vaccine development field has used singleor prime boost heterologous Envs as immunogens, but to date has notfound a regimen to induce high levels of bnAbs.

Recently, a new paradigm for design of strategies for induction ofbroadly neutralizing antibodies was introduced, that of B cell lineageimmunogen design (Nature Biotech. 30: 423, 2012) in which the inductionof bnAb lineages is recreated. It was recently demonstrated the power ofmapping the co-evolution of bnAbs and founder virus for elucidating theEnv evolution pathways that lead to bnAb induction (Nature 496: 469,2013). From this type of work has come the hypothesis that bnAbinduction will require a selection of antigens to recreate the “swarms”of sequentially evolved viruses that occur in the setting of bnAbgeneration in vivo in HIV infection (Nature 496: 469, 2013).

A critical question is why the CH505 immunogens are better than otherimmunogens. This rationale comes from three recent observations. First,a series of immunizations of single putatively “optimized” or “native”trimers when used as an immunogen have not induced bnAbs as singleimmunogens. Second, in all the chronically infected individuals who dodevelop bnAbs, they develop them in plasma after ˜2 years. When theseindividuals have been studied at the time soon after transmission, theydo not make bnAbs immediately.

Two other considerations are important. The first is that for the CH103bnAb CD4 binding site lineage, the VH4-59 and Vλ3-1 genes are common asare the VDJ, VJ recombinations of the lineage (Liao, Nature 496: 469,2013). In addition, the bnAb sites are so unusual, we are finding thatthe same VH and VL usage is recurring in multiple individuals. Thus, wecan expect the CH505 Envs to induce CD4 binding site antibodies in manydifferent individuals.

Finally, one needs to make a choice regarding gp120 vs. gp160 for thegenetic immunization However, in acute infection, gp41 non-neutralizingantibodies are dominant and overwhelm gp120 responses (Tomaras, G et al.J. Virol. 82: 12449, 2008; Liao, H X et al. JEM 208: 2237, 2011).Recently we have found that the HVTN 505 DNA prime, rAd5 vaccine trialthat utilized gp140 as an immunogen, also had the dominant response ofnon-neutralizing gp41 antibodies. Thus, the use of gp160 vs gp120 forgp41 dominance needs to be evaluated.

In certain aspects, the invention provides a strategy for induction ofbnAbs, which involves selecting and developing immunogens designed torecreate the antigenic evolution of Envs that occur when bnAbs developin the context of infection.

That broadly neutralizing antibodies (bnAbs) occur in nearly all serafrom chronically infected HIV-1 subjects suggests anyone can developsome bnAb response if exposed to immunogens via vaccination. Workingback from mature bnAbs through intermediates enabled understanding theirdevelopment from the unmutated ancestor, and showed that antigenicdiversity preceded the development of population breadth. See Liao etal. (2013) Nature 496, 469-476. In this study, an individual “CH505” wasfollowed from HIV-1 transmission to development of broadly neutralizingantibodies. This individual developed antibodies targeted to CD4 bindingsite on gp120. In this individual the virus was sequenced over time, andbroadly neutralizing antibody clonal lineage (“CH103”) was isolated byantigen-specific B cell sorts, memory B cell culture, and amplified byVH/VL next generation pyrosequencing. See Liao et al. (2013) Nature 496,469-476.

Further analysis of envelopes and antibodies from the CH505 individualindicated that a non-CH103 Lineage participates in driving CH103-BnAbinduction. For example V1 loop, V5 loop and CD4 binding site loopmutations escape from CH103 and are driven by CH103 lineage. Loop Dmutations enhanced neutralization by CH103 lineage and are driven byanother lineage. Transmitted/founder Env, or another early envelope forexample W004.03, and/or W004.26, triggers naïve B cell with CH103Unmutated Common Ancestor (UCA) which develop in to intermediateantibodies. Transmitted/founder Env, or another early envelope forexample W004.03, and/or W004.26, also triggers non-CH103 autologousneutralizing Abs that drive loop D mutations in Env that have enhancedbinding to intermediate and mature CH103 antibodies and drive remainderof the lineage.

The invention provides various methods to choose a subset of viralvariants, including but not limited to envelopes, to investigate therole of antigenic diversity in serial samples. In other aspects, theinvention provides compositions comprising viral variants, for examplebut not limited to envelopes, selected based on various criteria asdescribed herein to be used as immunogens.

In other aspects, the invention provides immunization strategies usingthe selections of immunogens to induce cross-reactive neutralizingantibodies. In certain aspects, the immunization strategies as describedherein are referred to as “swarm” immunizations to reflect that multipleenvelopes are used to induce immune responses. The multiple envelopes ina swarm could be combined in various immunization protocols of primingand boosting.

Sequences/Clones

Described herein are nucleic and amino acids sequences of HIV-1envelopes. In certain embodiments, the described HIV-1 envelopesequences are gp160s. In certain embodiments, the described HIV-1envelope sequences are gp120s. Other sequences, for example but notlimited to gp140s, both cleaved and uncleaved, gp140 Envs with thedeletion of the cleavage (C) site, fusion (F) and immunodominant (I)region in gp41—named as gp140ΔCFI, gp140 Envs with the deletion of onlythe cleavage (C) site and fusion (F) domain—named as gp140ΔCF, gp140Envs with the deletion of only the cleavage (C)—named gp140ΔC (See e.g.Liao et al. Virology 2006, 353, 268-282), gp145s, gp150s, gp41s, whichare readily derived from the nucleic acid and amino acid gp160sequences. In certain embodiments the nucleic acid sequences are codonoptimized for optimal expression in a host cell, for example a mammaliancell, a rBCG cell or any other suitable expression system.

In certain embodiments, the envelope design in accordance with thepresent invention involves deletion of residues (e.g., 5-11, 5, 6, 7, 8,9, 10, or 11 amino acids) at the N-terminus. For delta N-terminaldesign, amino acid residues ranging from 4 residues or even fewer to 14residues or even more are deleted. These residues are between thematuration (signal peptide, usually ending with CX, X can be any aminoacid) and “VPVXXXX . . . ”. In case of CH505 T/F Env as an example, 8amino acids (italicized and underlined in the below sequence) weredeleted: MRVMGIQRNYPQWWIWSMLGFWMLMICNGMWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQE . . . (rest of envelope sequence is indicatedas “ . . . ”). CH505 Envelopes with delta N-terminal design are referredto as D8 or ΔN8 or deltaN8. In other embodiments, the delta N-designdescribed for CH505 T/F envelope can be used to make delta N-designs ofother CH505 envelopes. In certain embodiments, the invention relatesgenerally to an immunogen, gp160, gp120 or gp140, without an N-terminalHerpes Simplex gD tag substituted for amino acids of the N-terminus ofgp120, with an HIV leader sequence (or other leader sequence), andwithout the original about 4 to about 25, for example 11, amino acids ofthe N-terminus of the envelope (e.g. gp120). See WO2013/006688, e.g. atpages 10-12, the contents of which publication is hereby incorporated byreference in its entirety.

The general strategy of deletion of N-terminal amino acids of envelopesresults in proteins, for example gp120s, expressed in mammalian cellsthat are primarily monomeric, as opposed to dimeric, and, therefore,solves the production and scalability problem of commercial gp120 Envvaccine production. In other embodiments, the amino acid deletions atthe N-terminus result in increased immunogenicity of the envelopes.

In certain embodiments, the invention provides envelope sequences, aminoacid sequences and the corresponding nucleic acids, and in which the V3loop is substituted with the following V3 loop sequenceTRPNNNTRKSIRIGPGQTFY ATGDIIGNIRQAH (SEQ. ID NO. 1). This substitution ofthe V3 loop reduced product cleavage and improves protein yield duringrecombinant protein production in CHO cells.

In certain embodiments, the CH505 envelopes will have added certainamino acids to enhance binding of various broad neutralizing antibodies.Such modifications could include but not limited to, mutations at W680Gor modification of glycan sites for enhanced neutralization.

In certain aspects, the invention provides composition and methods whichuse a selection of sequential CH505 Envs, as gp120s, gp 140s cleaved anduncleaved and gp160s, as proteins, DNAs, RNAs, or any combinationthereof, administered as primes and boosts to elicit immune response.Sequential CH505 Envs as proteins would be co-administered with nucleicacid vectors containing Envs to amplify antibody induction.

In certain embodiments the invention provides immunogens andcompositions which include immunogens as trimers. In certainembodiments, the immunogens include a trimerization domain which is notderived from the HIV-1 envelope. In certain embodiments, thetrimerization domain is GCN4 (See FIGS. 1 and 2B). In other embodimentsthe trimerization domain is CD40L. In other embodiments, the immunogensinclude CD40L domain (See FIGS. 1 and 2B).

HIV-1 gp120 Trimer Vaccine Immunogens (FIG. 1):HIV-1 Env gp120 GCN4 Trimer

HIV-1 Env gp120 GCN4 trimer is designed to be expressed as solublerecombinant trimeric HIV-1 gp120 protein. HIV-1 Env gp120 is mutatedfrom residue R to E at the cleavage site of HIV-1 gp120 at the residuepositions R503 and R511 (or any mutations at this region) to destroyedthe cleavage site, a 6-residue linker (GSGSGS) (SEQ. ID. NO. 2) (thelinker can be variations of 3-20 residues in length) is added to theC-terminal end of HIV-1 gp120 followed by addition of 33 amino acidresidues of GCN4 sequence (RMKQIEDKIEEILSKIYHIENEIARIKKLIGER) (SEQ IDNO. 3).

HIV-1 Env gp120 GCN4 CD40L Trimer:

In certain embodiments the trimer design includes an immuneco-stimulator. HIV-1 Env gp120 GCN4 CD40L trimer is designed to beexpressed as soluble recombinant trimeric HIV-1 gp120 proteinco-expressed with functional CD40L as immune co-stimulator. HIV-1 Envgp120 is mutated from residue R to E at the cleavage site of HIV-1 gp120at the residue positions R503 and R511 (or any mutations at this region)to destroyed the cleavage site, a 6-residue linker (GSGSGS) (SEQ. ID NO.2) (the linker can be variations of 3-20 residues in length) is added tothe C-terminal end of HIV-1 gp120, 33 amino acid residues of GCN4sequence (RMKQIEDKIEEILSKIYHIENEIARIKKLIGER) (SEQ ID NO 4) is added tothe C terminal end of the 6-residue linker, then a 11-residue liner(GGSGGSGGSGG) (SEQ ID NO. 5) (the linker can be variations of 3-20residues in length) is added to the C terminal end of the GCN4 domain,followed by addition of the sequence of the functional extracellulardomain of the human CD40 ligand (L) E113-L261.

HIV-1 Env gp120 GCN4 CD40L Trimer with His Tag:

HIV-1 Env gp120 GCN4 CD40L trimer with His tag is designed to beexpressed as soluble recombinant trimeric HIV-1 gp120 proteinco-expressed with functional CD40L as immune co-stimulator. HIV-1 Envgp120 is mutated from residue R to E at the cleavage site of HIV-1 gp120at the residue positions R503 and R511 (or any mutations at this region)to destroyed the cleavage site, a 6-residue linker (GSGSGS) (SEQ ID NO2)(the linker can be variations of 3-20 residues in length) is added tothe C-terminal end of HIV-1 gp120, 33 amino acid residues of GCN4sequence (RMKQIEDKIEEILSKIYHIENEIARIKKLIGER) (SEQ ID NO 6) is added tothe C terminal end of the 6-residue linker, a 11-residue liner(GGSGGSGGSGG) (SEQ ID NO 7) (the linker can be variations of 3-20residues in length) is added to the C terminal end of the GCN4 domain,then the sequence of the functional extracellular domain of the humanCD40 ligand (L) E113-L261 is then added followed by addition of 10histine residues as tag (the His tag can be more or less of 10residues). His-tag is added to anchor the HIV-1 gp120GCN4 CD40L intoliposome through nickel.

In non-limiting embodiments the liposome comprises cholesterol, viralmembrane lipid, anionic lipid, POPC(1-Palmitoyl-2-Oleoyl-sn-Giycero-3-Phosphocholine), POPE(1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine),1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS), DMPA(1,2-Dimyristoyl-sn-Glycero-3-Phosphate), DPPC, DPPG, Sphingomyelin, orany combination thereof. In certain embodiments the liposome comprisescholesterol. In certain embodiments the liposome comprises POPC. Incertain embodiments the liposome comprises phosphatidylserine. Incertain embodiments, the liposome comprises phosphatidic acid. Incertain embodiments, the liposome comprises cardiolipin. In certainembodiments the liposome comprises cholesterol, POPC, POPE, and DMPA.The specific composition and ratio of the lipids in the liposome can bedetermined experimentally, so long as the liposome composition retainsthe antigenic and/or immunogenic properties of the HIV envelope.Non-limiting examples of methods to determine these properties aredescribed herein.

Using the instant disclosure of envelope timers, any HIV-1 envelope canbe designed as a trimer. In certain embodiments the HIV-1 envelope isany one of the envelopes or selection of envelopes in ApplicationWO2014042669 (PCT/US PCT/US2013/000210), U.S. Application Ser. No.61/955,402 (“Swarm Immunization with Envelopes form CH505” Examples 2-4,FIGS. 14-19); US Application Ser. Nos. 61/972,531 and 62/027,427(Examples 2-3, FIGS. 18-24) the contents of which applications areherein incorporated by reference in their entirety.

In certain embodiments, the compositions and methods include anyimmunogenic HIV-1 sequences to give the best coverage for T cell helpand cytotoxic T cell induction. In certain embodiments, the compositionsand methods include mosaic and/or consensus HIV-1 genes to give the bestcoverage for T cell help and cytotoxic T cell induction. In certainembodiments, the compositions and methods include mosaic group M and/orconsensus genes to give the best coverage for T cell help and cytotoxicT cell induction. In some embodiments, the mosaic genes are any suitablegene from the HIV-1 genome. In some embodiments, the mosaic genes areEnv genes, Gag genes, Pol genes, Nef genes, or any combination thereof.See e.g. U.S. Pat. No. 7,951,377. In some embodiments the mosaic genesare bivalent mosaics. In some embodiments the mosaic genes aretrivalent. In some embodiments, the mosaic genes are administered in asuitable vector with each immunization with Env gene inserts in asuitable vector and/or as a protein. In some embodiments, the mosaicgenes, for example as bivalent mosaic Gag group M consensus genes, areadministered in a suitable vector, for example but not limited to HSV2,would be administered with each immunization with Env gene inserts in asuitable vector, for example but not limited to HSV-2.

In certain aspects the invention provides compositions and methods ofEnv genetic immunization either alone or with Env proteins to recreatethe swarms of evolved viruses that have led to bnAb induction.Nucleotide-based vaccines offer a flexible vector format to immunizeagainst virtually any protein antigen. Currently, two types of geneticvaccination are available for testing—DNAs and mRNAs.

In certain aspects the invention contemplates using immunogeniccompositions wherein immunogens are delivered as DNA. See Graham B S,Enama M E, Nason M C, Gordon I J, Peel S A, et al. (2013) DNA VaccineDelivered by a Needle-Free Injection Device Improves Potency of Primingfor Antibody and CD8+ T-Cell Responses after rAd5 Boost in a RandomizedClinical Trial. PLoS ONE 8(4): e59340, page 9. Various technologies fordelivery of nucleic acids, as DNA and/or RNA, so as to elicit immuneresponse, both T-cell and humoral responses, are known in the art andare under developments. In certain embodiments, DNA can be delivered asnaked DNA. In certain embodiments, DNA is formulated for delivery by agene gun. In certain embodiments, DNA is administered byelectroporation, or by a needle-free injection technologies, for examplebut not limited to Biojector® device. In certain embodiments, the DNA isinserted in vectors. The DNA is delivered using a suitable vector forexpression in mammalian cells. In certain embodiments the nucleic acidsencoding the envelopes are optimized for expression. In certainembodiments DNA is optimized, e.g. codon optimized, for expression. Incertain embodiments the nucleic acids are optimized for expression invectors and/or in mammalian cells. In non-limiting embodiments these arebacterially derived vectors, adenovirus based vectors, rAdenovirus(Barouch D H, et al. Nature Med. 16: 319-23, 2010), recombinantmycobacteria (i.e., rBCG or M smegmatis) (Yu, J S et al. ClinicalVaccine Immunol. 14: 886-093, 2007; ibid 13: 1204-11, 2006), andrecombinant vaccinia type of vectors (Santa S. Nature Med. 16: 324-8,2010), for example but not limited to ALVAC, replicating (Kibler K V etal., PLoS One 6: e25674, 2011 nov 9.) and non-replicating (Perreau M etal. J. virology 85: 9854-62, 2011) NYVAC, modified vaccinia Ankara(MVA)), adeno-associated virus, Venezuelan equine encephalitis (VEE)replicons, Herpes Simplex Virus vectors, and other suitable vectors.

In certain aspects the invention contemplates using immunogeniccompositions wherein immunogens are delivered as DNA or RNA in suitableformulations. Various technologies which contemplate using DNA or RNA,or may use complexes of nucleic acid molecules and other entities to beused in immunization. In certain embodiments, DNA or RNA is administeredas nanoparticles consisting of low dose antigen-encoding DNA formulatedwith a block copolymer (amphiphilic block copolymer 704). See Cany etal., Journal of Hepatology 2011 vol. 54 j 115-121; Arnaoty et al.,Chapter 17 in Yves Bigot (ed.), Mobile Genetic Elements: Protocols andGenomic Applications, Methods in Molecular Biology, vol. 859, pp293-305(2012); Arnaoty et al. (2013) Mol Genet Genomics. 2013 August;288(7-8):347-63. Nanocarrier technologies called Nanotaxi® forimmunogenic macromolecules (DNA, RNA, Protein) delivery are underdevelopment. Seewww.incellart.com/en/research-and-development/technologies.html.

In certain aspects the invention contemplates using immunogeniccompositions wherein immunogens are delivered as recombinant proteins.Various methods for production and purification of recombinant proteinssuitable for use in immunization are known in the art.

The immunogenic envelopes can also be administered as a protein boost incombination with a variety of nucleic acid envelope primes (e.g., HIV-1Envs delivered as DNA expressed in viral or bacterial vectors).

Dosing of proteins and nucleic acids can be readily determined by askilled artisan. A single dose of nucleic acid can range from a fewnanograms (ng) to a few micrograms GO or milligram of a singleimmunogenic nucleic acid. Recombinant protein dose can range from a fewμg micrograms to a few hundred micrograms, or milligrams of a singleimmunogenic polypeptide.

Administration: The compositions can be formulated with appropriatecarriers using known techniques to yield compositions suitable forvarious routes of administration. In certain embodiments thecompositions are delivered via intramascular (IM), via subcutaneous, viaintravenous, via nasal, via mucosal routes.

The compositions can be formulated with appropriate carriers andadjuvants using techniques to yield compositions suitable forimmunization. The compositions can include an adjuvant, such as, forexample but not limited to, alum, poly IC, MF-59 or other squalene-basedadjuvant, ASOIB, or other liposomal based adjuvant suitable for proteinor nucleic acid immunization. In certain embodiments, the adjuvant isGSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shownby GSK to be as potent as the similar adjuvant AS01B but to be lessreactogenic using HBsAg as vaccine antigen [Leroux-Roels et al., IABSConference, Apr. 2013, 9]. In certain embodiments, TLR agonists are usedas adjuvants. In some embodiments, the TLR agonist is a TLR4 agonist,such as but not limited to GLA/SE. In other embodiment, adjuvants whichbreak immune tolerance are included in the immunogenic compositions. Insome embodiments the adjuvant is TLR7 or a TLR7/8 agonist, or a TLR-9agonist, or a combination thereof. See PCT/US2013/029164.

There are various host mechanisms that control bNAbs. For example highlysomatically mutated antibodies become autoreactive and/or less fit(Immunity 8: 751, 1998; PloS Comp. Biol. 6 e1000800, 2010; J. Thoret.Biol. 164:37, 1993); Polyreactive/autoreactive naïve B cell receptors(unmutated common ancestors of clonal lineages) can lead to deletion ofAb precursors (Nature 373: 252, 1995; PNAS 107: 181, 2010; J. Immunol.187: 3785, 2011); Abs with long HCDR3 can be limited by tolerancedeletion (JI 162: 6060, 1999; JCI 108: 879, 2001). BnAb knock-in mousemodels are providing insights into the various mechanisms of tolerancecontrol of MPER BnAb induction (deletion, anergy, receptor editing).Other variations of tolerance control likely will be operative inlimiting BnAbs with long HCDR3s, high levels of somatic hypermutations.2F5 and 4E10 BnAbs were induced in mature antibody knock-in mouse modelswith MPER peptide-liposome-TLR immunogens. Next step is immunization ofgermline mouse models and humans with the same immunogens.

In certain embodiments the immunogens and compositions of the inventioncomprise immunostimulatory components. In a non-limiting embodiment, theimmunogen comprises a CD40L.

In certain embodiments, the compositions and methods comprise anysuitable agent or immune modulation which could modulate mechanisms ofhost immune tolerance and release of the induced antibodies. Innon-limiting embodiments modulation includes PD-1 blockade; T regulatorycell depletion; CD40L hyperstimulation; soluble antigen administration,wherein the soluble antigen is designed such that the soluble agenteliminates B cells targeting dominant epitopes, or a combinationthereof. In certain embodiments, an immunomodulatory agent isadministered in at time and in an amount sufficient for transientmodulation of the subject's immune response so as to induce an immuneresponse which comprises broad neutralizing antibodies against HIV-1envelope. Non-limiting examples of such agents is any one of the agentsdescribed herein: e.g. chloroquine (CQ), PTP1B Inhibitor—CAS765317-72-4—Calbiochem or MSI 1436 clodronate or any otherbisphosphonate; a Foxo1 inhibitor, e.g. 344355|Foxo1 Inhibitor,AS1842856—Calbiochem; Gleevac, anti-CD25 antibody, anti-CCR4 Ab, anagent which binds to a B cell receptor for a dominant HIV-1 envelopeepitope, or any combination thereof. In certain embodiments, the methodscomprise administering a second immunomodulatory agent, wherein thesecond and first immunomodulatory agents are different.

EXAMPLES Example 1: GCN4 Envelope Trimers and CD40L ContainingImmunogens Bind HIV-1 Envelope Antibodies and are Functionally Active

Provided is one example of the design and formulation of liposomes thatpresent immune-modulating CD40 ligand (CD40L) and HIV-1 gp41neutralizing antigen. CD40L, the ligand for CD40 expressed on B-cellsurface is anchored on the liposomes that had HIV-1 gp41 MPER peptideimmunogen conjugated in them. Two broadly neutralizing gp41 membraneproximal external region (MPER) antibodies (2F5, 4E10) bound strongly toCD40L conjugated MPER peptide liposomes. This construct has importantapplication as an experimental AIDS vaccine in providingimmune-modulating effect to stimulate proliferation of B-cells capableof producing neutralizing antibodies targeting HIV-1 gp41 MPER region.

CD40L-gp41 MPER peptide-liposome conjugates: Recombinant CD40L with anN-terminal Histidine Tag

(SEQ ID NO 8) (MGSSHHHHHH SSGLVPRGSH MQKGDQNPQIAAHVISEASS KTTSVLQWAE KGYYTMSNNL VTLENGKQLTVKRQGLYYIY AQVTFCSNRE ASSQAPFIAS LCLKSPGRFERILLRAANTH SSAKPCGQQS IHLGGVFELQ PGASVFVNVT DPSQVSHGTG FTSFGLLKL) was anchored to MPER peptide liposomes via His-Ni-NTA chelation bymixing CD40L with MPER656-Ni-NTA liposomes at 1:50 CD40L and Ni-NTAmolar ratio (Figure-12).

The construction of MPER peptide Ni-NTA liposomes utilized the method ofco-solubilization of MPER peptide having a membrane anchoring amino acidsequence and synthetic lipids1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC),1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE),1,2-Dimyristoyl-sn-Glycero-3-Phosphate (DMPA), Cholesterol and1,2-dioleoyl-sn-Glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiaceticacid)succinyl] (nickel salt) (DGS-NTA(Ni) at mole fractions 0.216,35.00, 25.00, 20.00, 1.33 and 10 respectively. Appropriate amount ofMPER peptide dissolved in chloroform-methanol mixture (7:3 v/v),appropriate amounts of chloroform stocks of phospholipids were dried ina stream of nitrogen followed by overnight vacuum drying. Liposomes weremade from the dried peptide-lipid film in phosphate buffered saline (pH7.4) using extrusion technology.

Biolayer interferometry (BLI) assay showed the binding of anti-humanCD40L antibody to CD40L-MPER656 liposomes and confirmed the correctpresentation of CD40 L on liposome surface (FIG. 13A). The broadlyneutralizing HIV-1 gp41 MPER antibodies 2F5 and 4E10 bound strongly toCD40L-MPER656 liposomes (FIGS. 13B-C) and demonstrated that the CD40Lco-display did not impede the presentation of the epitopes of 2F5 and4E10 mAbs.

FIGS. 14 and 15 show that CD40L containing immunogens activate humanCD40 expressing HEK cells.

Example 2—Combination of Antigens from CH505 Envelope Sequences forImmunization

Provided herein are non-limiting examples of combinations of antigensderived from CH505 envelope sequences for a swarm immunization. Theselection includes priming with a virus which binds to the UCA, forexample a T/F virus or another early (e.g. but not limited to week004.3, or 004.26) virus envelope. In certain embodiments the prime couldinclude D-loop variants. In certain embodiments the boost could includeD-loop variants.

Non-limiting embodiments of envelopes selected for swarm vaccination areshown as the selections described below. A skilled artisan wouldappreciate that a vaccination protocol can include a sequentialimmunization starting with the “prime” envelope(s) and followed bysequential boosts, which include individual envelopes or combination ofenvelopes. In another vaccination protocol, the sequential immunizationstarts with the “prime” envelope(s) and is followed with boosts ofcumulative prime and/or boost envelopes. In certain embodiments, theprime does not include T/F sequence (W000.TF). In certain embodiments,the prime includes w004.03 envelope. In certain embodiments, the primeincludes w004.26 envelope. In certain embodiments, the immunizationmethods do not include immunization with HIV-1 envelope T/F. In otherembodiments for example the T/F envelope may not be included whenw004.03 or w004.26 envelope is included. In certain embodiments, thereis some variance in the immunization regimen; in some embodiments, theselection of HIV-1 envelopes may be grouped in various combinations ofprimes and boosts, either as nucleic acids, proteins, or combinationsthereof.

In certain embodiments the immunization includes a prime administered asDNA, and MVA boosts. See Goepfert, et al. 2014; “Specificity and 6-MonthDurability of Immune Responses Induced by DNA and Recombinant ModifiedVaccinia Ankara Vaccines Expressing HIV-1 Virus-Like Particles” J InfectDis. 2014 Feb. 9. [Epub ahead of print].

HIV-1 Envelope selection A (ten envelopes: sensitive envelopes):703010505.TF, 703010505.W4.03, 703010505.W4.26, 703010505.W14.21,703010505.W20.14, 703010505.W30.28, 703010505.W30.13, 703010505.W53.31,703010505.W78.15, 703010505.W100.B4, optionally in certain embodimentsdesigned as trimers. See U.S. Provisional Application No. 62/027,427incorporated by reference.

HIV-1 Envelope selection B (twenty envelopes: sensitive envelopes):703010505.TF, 703010505.W4.03, 703010505.W4.26, 703010505.W14.3,703010505.W14.8, 703010505.W14.21, 703010505.W20.7, 703010505.W20.26,703010505.W20.9, 703010505.W20.14, 703010505.W30.28, 703010505.W30.12,703010505.W30.19, 703010505.W30.13, 703010505.W53.19, 703010505.W53.13,703010505.W53.31, 703010505.W78.1, 703010505.W78.15, 703010505.W100.B4,optionally in certain embodiments designed as trimers. See U.S.Provisional Application No. 62/027,427 incorporated by reference.

HIV-1 Envelope selection C (four envelopes): 703010505.TF,703010505.W53.16, 703010505.W78. 33, 703010505.W100.B6, optionally incertain embodiments designed as trimers. See WO2014042669.

HIV-1 Envelope selection D (ten production envelopes): CH505.M6;CH505.M11; CH505w020.14; CH505w030.28; CH505w030.21; CH505w053.16;CH505w053.31; CH505w078.33; CH505w078.15; CH505w100.B6, optionally incertain embodiments designed as trimers. See FIG. 17.

HIV-1 Envelopes selection E (ten early envelopes): optionally in certainembodiments designed as trimers. CH505.M11; CH505.w004.03;CH505.w020.14; CH505.w030.28; CH505.w030.12; CH505.w020.2;CH505.w030.10; CH505.w078.15; CH505.w030.19; CH505.w030.21, optionallyin certain embodiments designed as trimers. See FIG. 24.

HIV-1 Envelope selection F (ten production envelopes (10PR)): CH505.T/F;CH505.M11; CH505w020.14; CH505w030.28; CH505w030.21; CH505w053.16;CH505w053.31; CH505w078.33; CH505w078.15; CH505w100.B6, optionally incertain embodiments designed as trimers. See FIG. 18B.

Example 3: Examples of Immunization Protocols in Subjects with Swarms ofHIV-1 Envelopes

Immunization protocols contemplated by the invention include envelopesequences as described herein including but not limited to nucleic acidsand/or amino acid sequences of gp160s, gp150s, cleaved and uncleavedgp140s, gp120s, gp41s, N-terminal deletion variants as described herein,cleavage resistant variants as described herein, or codon optimizedsequences thereof. A skilled artisan can readily modify the gp160 andgp120 sequences described herein to obtain these envelope variants. Theswarm immunization protocols can be administered in any subject, forexample monkeys, mice, guinea pigs, or human subjects. The swarmimmunization protocols include additive and/or sequential immunizationwith the selections of HIV envelopes.

In non-limiting embodiments, the immunization includes a nucleic acid isadministered as DNA, for example in a modified vaccinia vector (MVA). Innon-limiting embodiments, the nucleic acids encode gp160 envelopes. Inother embodiments, the nucleic acids encode gp120 envelopes. In otherembodiments, the boost comprises a recombinant gp120 envelope. Thevaccination protocols include envelopes formulated in a suitable carrierand/or adjuvant, for example but not limited to alum. In certainembodiments the immunizations include a prime, as a nucleic acid or arecombinant protein, followed by a boost, as a nucleic acid or arecombinant protein. A skilled artisan can readily determine the numberof boosts and intervals between boosts.

In non-limiting embodiments, the prime includes a 703010505.TF envelopeand a loop D variant as described herein. In non-limiting embodiments,the prime includes a 703010505.TF envelope and/or 703010505.W4.03,703010505.W4.26 envelope, and a loop D variant as described herein. Incertain embodiments, the loop D variant is M6. In certain embodiments,the loop D variant is M5. In certain embodiments, the loop D variant isM10. In certain embodiments, the loop D variant is M19. In certainembodiments, the loop D variant is M11. In certain embodiments, the loopD variant is M20. In certain embodiments, the loop D variant is M21. Incertain embodiments, the loop D variant is M9. In certain embodiments,the loop D variant is M8. In certain embodiments, the loop D variant isM7.

Table 1 shows a non-limiting example of a sequential immunizationprotocol using a swarm of HIV1 envelopes (703010505.TF, 703010505.W4.03,703010505.W4.26, 703010505.W14.21, 703010505.W20.14, 703010505.W30.28,703010505.W30.13, 703010505.W53.31, 703010505.W78.15, 703010505.W100.B4,optionally in certain embodiments designed as trimers. In a non-limitingembodiment, a suggested grouping for prime and boost is to begin withthe CH505 TF+W4.03, then boost with a mixture of w4.26+14.21+20.14, thenboost with a mixture of w30.28+30.13+53.31, then boost with a mixture ofw78.15+100.B4.

Envelope Prime Boost(s) Boost(s) Boost(s) CH505 TF + CH505 TF + W4.03W4.03 as a nucleic acid e.g. DNA/MVA vector and/or protein w4.26 +w4.26 + 14.21 + 14.21 + 20.14 20.14 as a nucleic acid e.g. DNA/MVAvector and/or protein w30.28 + w30.28 + 30.13 + 30.13 + 53.31 53.31 as anucleic acid e.g. DNA/MVA vector and/or protein w78.15 + w78.15 + 100.B4100.B4 as a nucleic acid e.g. DNA/MVA vector and/or protein

A skilled artisan can readily determine the number and interval betweenboosts.

Table 2 shows a non-limiting example of a sequential immunizationprotocol using a swarm of HIV1 envelopes optionally in certainembodiments designed as trimers.

Envelope Prime Boost(s) 703010505.TF, 703010505.TF 703010505.TF,703010505.W4.03, (optionally 703010505.W4.03, 703010505.W4.26,703010505.W4.03, 703010505.W4.26, 703010505.W14.21, 703010505.W4.26)703010505.W14.21, 703010505.W20.14, as a nucleic acid 703010505.W20.14,703010505.W30.28, e.g. DNA/MVA 703010505.W30.28, 703010505.W30.13,vector and/or 703010505.W30.13, 703010505.W53.31, protein703010505.W53.31, 703010505.W78.15, 703010505.W78.15, 703010505.W100.B4.703010505.W100.B4 as a nucleic acid e.g. DNA/MVA vector and/or protein

A skilled artisan can readily determine the number and interval betweenboosts.

For a 20mer immunization regimen (envelopes (703010505.TF,703010505.W4.03, 703010505.W4.26, 703010505.W14.3, 703010505.W14.8,703010505.W14.21, 703010505.W20.7, 703010505.W20.26, 703010505.W20.9,703010505.W20.14, 703010505.W30.28, 703010505.W30.12, 703010505.W30.19,703010505.W30.13, 703010505.W53.19, 703010505.W53.13, 703010505.W53.31,703010505.W78.1, 703010505.W78.15, 703010505.W100.B4), in a non-limitingembodiment, one can prime with CH505 TF+W4.03, then boost with a mixtureof w4.26+14.21+20.14+14.3+14.8+20.7, then boost with a mixture of w20.26+20.9+30.12+w30.28+30.13+53.31, then boost with a mixture ofw78.15+100.B4+30.19+53.19+53.13+78.1. Other combinations of envelopesare contemplated for boosts.

Table 3 shows a non-limiting example of a sequential immunizationprotocol using a swarm of HIV1 envelopes optionally in certainembodiments designed as trimers

Envelope Prime Boost(s) 703010505.TF, 703010505.TF, 703010505.TF,703010505.W4.03, (optionally 703010505.W4.03, 703010505.W4.26,703010505.W4.03, 703010505.W4.26, 703010505.W14.3, 703010505.W4.26,703010505.W14.3, 703010505.W14.8, 703010505.W14.3, 703010505.W14.8,703010505.W14.21, 703010505.W14.8, 703010505.W14.21, 703010505.W20.7,703010505.W14.21), 703010505.W20.7, 703010505.W20.26, as a nucleic acid703010505.W20.26, 703010505.W20.9, e.g. DNA/MVA 703010505.W20.9,703010505.W20.14, vector and/or 703010505.W20.14, 703010505.W30.28,protein 703010505.W30.28, 703010505.W30.12, 703010505.W30.12,703010505.W30.19, 703010505.W30.19, 703010505.W30.13, 703010505.W30.13,703010505.W53.19, 703010505.W53.19, 703010505.W53.13, 703010505.W53.13,703010505.W53.31, 703010505.W53.31, 703010505.W78.1, 703010505.W78.1,703010505.W78.15, 703010505.W78.15, 703010505.W100.B4.703010505.W100.B4. as a nucleic acid e.g. DNA/MVA vector and/or protein

A skilled artisan can readily determine the number and interval betweenboosts.

Table 4 shows a non-limiting example of a sequential immunizationprotocol using a swarm of HIV 1 envelopes optionally in certainembodiments designed as trimers.

Envelope Prime Boost(s) Boost(s) Boost(s) CH505.M6 CH505.M6 CH505.M11CH505.M11 as a nucleic acid e.g. DNA/MVA vector and/or proteinCH505w020.14 CH505w020.14 CH505w030.28 CH505w030.28 as a nucleic acide.g. DNA/MVA vector and/or protein CH505w078.15 CH505w078.15CH505w053.31 CH505w053.31 CH505w030.21 CH505w030.21as a nucleic acide.g. DNA/MVA vector and/or protein CH505w078.33 CH505w078.33CH505w053.16 CH505w053.16 CH505w100.B6 CH505w100.B6 as a nucleic acide.g. DNA/MVA vector and/or protein

A skilled artisan can readily determine the number and interval betweenboosts.

Table 5 shows a non-limiting example of a sequential immunizationprotocol using a swarm of HIV 1 envelopes optionally in certainembodiments designed as trimers.

Envelope Prime Boost(s) Boost(s) Boost(s) CH505.T/F CH505.T/F CH505.M11CH505.M11 as a nucleic acid e.g. DNA/MVA vector and/or proteinCH505w020.14 CH505w020.14 CH505w030.28 CH505w030.28 as a nucleic acide.g. DNA/MVA vector and/or protein CH505w078.15 CH505w078.15CH505w053.31 CH505w053.31 CH505w030.21 CH505w030.21as a nucleic acide.g. DNA/MVA vector and/or protein CH505w078.33 CH505w078.33CH505w053.16 CH505w051.36 CH505w100.B6 CH505w100.B6 as a nucleic acide.g. DNA/MVA vector and/or protein

A skilled artisan can readily determine the number and interval betweenboosts.

Example 4: Selection of Ten Early Envelopes

Provided is the approach to selecting a 10-immunogen set from CH505 (SeeFIG. 24). Here we choose 10 low-diversity variants from the subjectearly on, rather than down-selecting from a short list of 18, (which youare already making) to represent diversity that appeared through week160, and includes samples after escape from the mature CH103 mAb.

Without being bound by theory, the hypothesis is that affinitymaturation in the presence of antigenic diversity helps select forbreadth, allowing it to evolve gradually from a population of Envsselected by clonal autologous neutralization response. But here we wouldtest whether modest variation in the antigen might better stimulateresponses that allow the clonal lineage to interact and adapt, while thefull range of variation might introduce too much diversity for thedeveloping lineage. For example, a set of Envs with 1 or 2 substitutionsin an epitope might reduce affinity, but still allow binding, and theevolving B cell population would be able to adapt. Such variants mightallow more “generalists” to evolve. Env variants fully escaped fromearly lineage clones might be immunologically silent, and less able todraw increased breadth from the B cell clones.

This is essentially like trying a serial version of the swarm vaccine of100, where we plan on starting with the low-diversity forms, andincrease diversity as we vaccinate, but by making these 10 we could tryother delivery strategies.

We selected a set of 10 gp120s for use as candidate immunogens. Thefocus here is on Env diversity at week 30, which coincides with anexpansion in heterologous neutralization seen also by antigeniccartography. Unlike the TF and earlier forms, all week 30 sequencescontain the V3 glycan shift from 334 to 332.

We identified Env sites to use as criteria for Env selection. The siteswere determined by TF loss, neutralization signatures, and contact withthe CD4bs and CH103 bnAb (Table 6): (a) At least 80% TF loss throughweek 160 yielded 36 sites, as described previously. (b) Neutralizationsignatures for single or PNG sites with q<0.1 for tree-correctedsignatures of IC50s below 20 μg/ml, as described previously. (c) Thelist of contact sites was expanded by one amino acid up- and downstreamof each known contact, to include a slightly larger neighborhood ofcontact sites. These 66 HXB2 sites grew to 71 sites when mapped onto theCH505 Env alignment. When reviewed for polymorphisms, 28 of these sitesvary in CH505 over the sampling period.

CTL responses were mapped and found one ELISpot positive peptide on theC-terminus of the V4 loop, sites 409-418, EGSDTITLPC in HXB2, NSTRTITIHCin CH505. CTL epitope variants are identified among selected sites inTable 5.

Neutralization sensitivity of autologous Envs to mAbs in the CH103lineage further informs selection of 10 Envs (FIG. 19). Comparingselected Envs with concatenated sites (FIG. 20) allows selection forincremental progression of mAb sensitivities (FIG. 20D). An abrupttransition between neutralization sensitivity to IA7 and IA3 limitsavailable Envs from week 30 (FIG. 19), perhaps because of the mAbdiscontinuity induced by a shift in light-chain usage from UCA to IA2light chain associations with IA4 and IA3 heavy chains, respectively(i.e. IA4 mAb is 14 VH and UCA VL; IA3 mAb is VH 13 with VL 12).

CH505 Env diversity and neutralization to the CH103 lineage mAbs,together with the distributions of proposed sets of 4, 10 (new and inpreparation), and 100 antigens are all compared by established methodsin FIG. 21.

TABLE 6 Alignment columns in Env “hot-spot” concatamer summaries. ColHXB2 AA CH505 Feature a: 36 sites with TF loss >80% 1 279 D N Loop D 2281 A V Loop D 3 332 O N PGT121 4 334 S O 2G12 5  144+ — — V1 6  144+ —— V1 7  144+ — — V1 8 413 T T V4/CTL 9 465 S — V5 10 464 E — V5 11 417 PH V4/CTL 12 330 H Y V3 13 300 N N V3 14 234 O T 8ANC195 15 302 N K V3 16756 I V gp41 17  463+ — — V5 18 398 S O V4 19 133 D O V1 20 460 N K V521 347 S K 22 275 V E Loop D 23 151 K I V1 24 356 O H 25 471 G G beta2426 147 M O V1 27 640 S E gp41 28 462 N N V5 29 145 G A V1 30 130 K O 31132 T T V1 32 620 E G gp41 33  4 K M SignalPep 34 325 N D V3 35 185 D DV2 36 412 D R V4/CTL b: 28 signature sites, q < 0.1 1 130 K O 2 132 T TV1 3 133 D O V1 4 135 K T V1 5 137 D — V1 6 146 R S V1 7 148 I S V1 8147 M O V1 9 149 M S V1 10 151 K I V1 11 160 O O PG9 12 200 V V 13 234 OT 8ANC195 14 328 Q E V3 15 332 O N PGT121 16 334 S O 2G12 17 336 A S 18347 S K 19 356 O H 20 358 T O 21 360 I T 22 416 L I V4/CTL 23 460 N K V524 461 S O V5 25 463 O T V5 26 743 D O Kennedy 27 745 S S Epitope 28 831E E LLP-1 c: 28 varying contacts 1 127 V V CD4 2 128 S T CD4 3 255 V V 4278 T T 5 279 D N Loop D 6 280 N N Loop D 7 281 A V Loop D 8 282 K KLoop D 9 283 T T Loop D 10 363 Q P 11 365 S S 12 367 G G 13 369 P L CD414 371 I I CD4 15 372 V T 16 424 I I 17 433 A A 18 460 N K V5 19 461 S OV5 20 462 N N V5 21 463 N T V5 22  463+ — — V5 23  463+ — — V5 24  463+— — V5 25  463+ — — V5 26  463+ — — V5 27  464+ E — V5 28 471 G G Beta24

Example 5: Non-Human Primate Studies

NHP 79: CH505T/F gp120 envelope in GLA/SE. NHP 85: CH505T/F gp140envelope in GLA/SE. This compares gp140 with gp120 induced antibodies.

NHP study of CH505T/F gp120 with GCN4 CH505 T/F in GLA/SE.

NHP study of CH505T/F gp120 with GCN4 CD40L CH505 T/F in GLA/SE.

NHP study of CH505T/F gp120 with GCN4 CD40L CH505 T/F in ALUM.

NHP study of CH505 T/F gp120 with GCN4 CD40L CH505 T/F=-HIS tag withliposomes in ALUM.

NHP study of M6 then rest of production 10 (Table 4) gp120 in sequencegp120 GNC4 CD40L CH505 trimers with ALUM or GLA/SE (depends onantigenicity).

NHP study of M6 then rest of production 10 (Table 4) gp120 in sequencegp120 GNC4 CD40L CH505 trimers in ALUM or GLA/SE (depends onantigenicity), with a dose of chloloquine orally each day 10 days beforeeach immunization and then a dose of CD25 Ab 5 days after eachimmunization. See U.S. Application Ser. No. 62/056,583 (“Tolerance”filed concurrently), which contents is herein incorporated by referencein its entirety.

The contents of all documents and other information sources cited hereinare herein incorporated by reference in their entirety.

Provided below are examples of sequences and HIV-1 envelopes disclosedin this application.

HIV-1 Envelope Selection D: Ten Production Envelopes

TABLE 4 FIG. 17 CH505.M6 CH505.M6D8gp120 (aa SEQ ID NO 9; nt SEQ ID NO:10), CH505.M6gp145 (nt SEQ ID NO 11), CH505.M6 gp160 (aa SEQ ID NO: 12;nt SEQ ID NO 13) CH505.M11 CH505.M11D8gp120 (aa SEQ ID NO 14; nt SEQ IDNO 15), CH505.M11gp145 (nt SEQ ID NO 16), CH505.M11 gp160 (aa SEQ ID NO17; nt SEQ ID NO 18) CH505w020.14 CH505w020.14D8gp120 (aa SEQ ID NO 19;nt SEQ ID NO: 20), CH505w020.14gp145 (nt SEQ ID NO 21), CH505w020.14gp160 (aa SEQ ID NO 22; nt SEQ ID NO 23) CH505w030.28CH505w030.28D8gp120 (aa SEQ ID NO 24; nt SEQ ID NO 25),CH505w030.28gp145(nt SEQ ID NO 26),, CH505w030.28 gp160 (aa SEQ ID NO27; nt SEQ ID NO 28) CH505w078.15 CH505w078.15D8gp120 (aa SEQ ID NO 29;nt SEQ ID NO 30), CH505w078.15gp145 (nt SEQ ID NO 40), CH505w078.15gp160 (aa SEQ ID NO 41; nt SEQ ID NO 42) CH505w053.31CH505w053.31D8gp120 (aa SEQ ID NO 43; nt SEQ ID NO 44),CH505w053.31gp145 (nt SEQ ID NO 45), CH505w053.31 gp160 (aa SEQ ID NO46; nt SEQ ID NO 47) CH505w030.21 CH505w030.21D8gp120 (aa SEQ ID NO 48;nt SEQ ID NO 49), CH505w30.21gp145 (nt SEQ ID NO 50), CH505w030.21 gp160(aa SEQ ID NO 51 nt SEQ ID NO 52) CH505w078.33 CH505w78.33gp120 (aa SEQID NO 54; nt SEQ ID NO 55), CH505w78.33gp145 (nt SEQ ID NO 56),CH505w078.33 gp160 (aa SEQ ID NO 57; nt SEQ ID NO 58) CH505w053.16CH505w053.16D8gp120 (aa SEQ ID NO 59; nt SEQ ID NO 60), CH505w53.16gp145(nt SEQ ID NO 61), CH505w053.16 gp160 (aa SEQ ID NO 62; nt SEQ ID NO 63)CH505w100.B6 CH505w100.B6D8gp120 (aa SEQ ID NO 64; nt SEQ ID NO 65),CH505w100.B6gp145 (nt SEQ ID NO 66), CH505w100.B6 gp160 (aa SEQ ID NO67; nt SEQ ID NO 68)

HIV-1 Envelopes Selection E: Ten Early Envelopes (FIG. 24)

FIG. 24 HIV-1 Envelopes Selection E CH505M11gp160 (aa SEQ ID NO 69)CH505.M11 CH505w004.03gp160 (aa SEQ ID NO 70) CH505.w004.03CH505w020.14gp160 (aa SEQ ID NO 71) CH505.w020.14 CH505w030.28gp160 (aaSEQ ID NO 72) CH505.w030.28 CH505w30.12gp160 (aa SEQ ID NO 73)CH505.w030.12 CH505w020.2gp160 (aa SEQ ID NO 74) CH505.w020.2CH505w030.10gp160 (aa SEQ ID NO 75) CH505.w030.10 CH505w078.15gp160 (aaSEQ ID NO 76) CH505.w078.15 CH505w030.19gp160 (aa SEQ ID NO 77)CH505.w030.19 CH505w030.21gp160 (aa SEQ ID NO 78) CH505.w030.21

HIV-1 Envelopes Selection C: Four Envelopes (FIG. 22)

FIG. 22 HIV-1 Envelopes Selection C CH505w000.TFgp160 (aa SEQ ID NO 79)703010505.TF, CH505w053.16gp160 (aa SEQ ID NO 80) 703010505.W53.16CH505w078.33gp160 (aa SEQ ID NO 81) 703010505.W78.33 CH505w100.B6gp160(aa SEQ ID NO 82) 703010505.W100.B6

HIV-1 Envelopes Selection F: Ten Production Envelopes (10PR)

TABLE 5 FIG. 17 CH505.T/F CH505w000.TFgp160 (aa SEQ ID NO 79) from FIG.22 CH505.M11 CH505.M11D8gp120 (aa SEQ ID NO 14; nt SEQ ID NO 15),CH505.M11gp145 (nt SEQ ID NO 16), CH505.M11 gp160 (aa SEQ ID NO 17; ntSEQ ID NO 18) CH505w020.14 CH505w020.14D8gp120 (aa SEQ ID NO 19; nt SEQID NO: 20), CH505w020.14gp145 (nt SEQ ID NO 21), CH505w020.14 gp160 (aaSEQ ID NO 22; nt SEQ ID NO 23) CH505w030.28 CH505w030.28D8gp120 (aa SEQID NO 24; nt SEQ ID NO 25), CH505w030.28gp145(nt SEQ ID NO 26),,CH505w030.28 gp160 (aa SEQ ID NO 27; nt SEQ ID NO 28) CH505w078.15CH505w078.15D8gp120 (aa SEQ ID NO 29; nt SEQ ID NO 30),CH505w078.15gp145 (nt SEQ ID NO 40), CH505w078.15 gp160 (aa SEQ ID NO41; nt SEQ ID NO 42) CH505w053.31 CH505w053.31D8gp120 (aa SEQ ID NO 43;nt SEQ ID NO 44), CH505w053.31gp145 (nt SEQ ID NO 45), CH505w053.31gp160 (aa SEQ ID NO 46; nt SEQ ID NO 47) CH505w030.21CH505w030.21D8gp120 (aa SEQ ID NO 48; nt SEQ ID NO 49), CH505w30.21gp145(nt SEQ ID NO 50), CH505w030.21 gp160 (aa SEQ ID NO 51; nt SEQ ID NO 52)CH505w078.33 CH505w78.33gp120 (aa SEQ ID NO 54; nt SEQ ID NO 55),CH505w78.33gp145 (nt SEQ ID NO 56), CH505w078.33 gp160 (aa SEQ ID NO 57;nt SEQ ID NO 58) CH505w053.16 CH505w053.16D8gp120 (aa SEQ ID NO 59; ntSEQ ID NO 60), CH505w53.16gp145 (nt SEQ ID NO 61), CH505w053.16 gp160(aa SEQ ID NO 62; nt SEQ ID NO 63) CH505w100.B6 CH505w100.B6D8gp120 (aaSEQ ID NO 64; nt SEQ ID NO 65), CH505w100.B6gp145 (nt SEQ ID NO 66),CH505w100.B6 gp160 (aa SEQ ID NO 67; nt SEQ ID NO 68)

What is claimed is:
 1. A composition comprising any one of the HIV-1envelope polypeptides corresponding to the HIV-1 envelopes CH505.M6,CH505.M11, CH505w020.14, CH505w030.28, CH505w078.15, CH505w053.31,CH505w030.21, CH505w078.33, CH505w053.16, CH505w100.B6 or a combinationthereof, or polynucleotide encoding the same.
 2. A compositioncomprising any one of the HIV-1 envelope polypeptides corresponding tothe HIV-1 envelopes CH505.T/F; CH505.M11; CH505w020.14; CH505w030.28;CH505w030.21; CH505w053.16; CH505w053.31; CH505w078.33; CH505w078.15;CH505w100.B6 or a combination thereof, or a polynucleotide encoding thesame.
 3. A composition comprising any one of the HIV-1 envelopepolypeptides corresponding to the HIV-1 envelopes CH505.M11,CH505.w004.03, CH505.w020.14, CH505.w030.28, CH505.w030.12,CH505.w020.2, CH505.w030.10, CH505.w078.15, CH505.w030.19 CH505.w030.21or a combination thereof, or polynucleotide encoding the same.
 4. Thecomposition of claims 1-3, wherein each HIV-1 envelope polypeptidecomprises polypeptide which is a gp 41, gp 120, gp 145, gp 150 or gp 160variant.
 5. The composition of claims 1-4, wherein the HIV-1 envelopesfurther comprise a peptide or polynucleotides corresponding to atrimerization domain selected from a group consisting GCN4 and CD40L. 6.The composition of claim 5, wherein the trimerization domain is linkedto the envelope sequence by an amino acid linker 3-20 amino acids long.7. A composition comprising MPER-peptide-liposome-CD40L conjugate. 8.The composition of claim 7, further comprising an N-terminal histonetag.
 9. The composition of any one of claims 1-8 further comprising anadjuvant.
 10. A method of inducing an immune response in a subjectcomprising administering the composition of any one of claims 1-9 in anamount sufficient to induce an immune response.
 11. The method of claim10, further comprising administering chloloquine before eachimmunization.
 12. The method of claim 10, further comprisingadministering anti-CD25 antibody before each immunization.
 13. Themethod of claim 10, further comprising administering anti-CD25 antibodyafter each immunization.
 14. The method of claim 10, wherein thecomposition comprises a nucleic acid, a protein or any combinationthereof.
 15. The method of claim 14, wherein the nucleic acid encodingthe envelope is operably linked to a promoter inserted in an expressionvector.
 16. The method of claim 14, wherein the protein is recombinant.17. The method of claim 14, wherein the composition is administered as aprime, a boost, or both.
 18. The method of claim 14, wherein thecomposition is administered as a multiple boosts.